We have discovered a 9.4 kb intervening sequence in the majority of DNA sequences coding for 28S ribosomal (rDNA) in Drosophila virilis; the position of the "interruption" is similar to that of perturbations of the same gene in D. melanogaster. We have also determined that the rDNA gene interruptions in these two distantly related species contain homologous sequences and the homology extends to putative rDNA interruptions in far more divergent species in the order Diptera. Sequences in the rDNA interruptions of Drosophila species have homologues elsewhere in the respective genomes, and interspecific homology extends to some of these as well. Our research aims are (1) to further characterize rDNA interruptions by restriction mapping and direct nucleotide sequencing, with emphasis being given to cloned portions of the D. virilis interruption that exhibit homology with non-rDNA sequences in the Drosophila genome, and portions that are homologous between species, and (2) to analyze in detail properties of cloned non-rDNA segments of the Drosophila genome that contain sequences homologous with rDNA interruptions. cDNA representing mRNAs will be cloned, and those that hybridize with the aforementioned cloned genome segments will be used as probes in the mapping of relative positions of coding and "interruption" sequences. Mapped restriction segments of clones will be hybridized with heterogenous nuclear RNA to determmine if "interruption" sequences are transcribed but subject to processing. Studies on rDNA interruptions in diverse species should enable us to discern patterns of the origin and evolution of rDNA intervening sequences and their non-rDNA homologues. Studies on the homologous sequences in non-rDNA portions of the genome are directed to the determination of physical and functional relationships between these and neighboring sequences, particularly those of the latter that code for messenger RNA.